JP5563475B2 - PEG interferon-beta preparation - Google Patents

Abstract

The invention relates to a liquid pharmaceutical composition comprising a pegylated IFN-β (PEG-IFN-β), an excipient, a surfactant and a buffer wherein said excipient is a polyol, wherein said surfactant is a non-ionic surfactant and wherein said buffer is a sodium acetate buffer.

Description

  The present invention relates to formulations of pegylated interferon beta (PEG-IFN-β).

  Interferon-β is a protein and is now known as a useful pharmaceutical applied for example in the treatment of multiple sclerosis (MS).

  For example, proteins can be altered in their sequence or by other modifications to change or improve their activity or stability. One such modification is the introduction of a bond to a polymer such as polyethylene glycol (PEG).

  Interferon-β conjugated with polyethylene glycol (PEG-IFN-β) is described, for example, in WO 99/55377 and EP 0593868 A1.

  Various attempts have been made in the medical field to stabilize proteins by lyophilization or in the form of liquid pharmaceutical compositions. The lyophilized material must be returned in solution prior to use. Concentrated or ready-made liquid pharmaceutical compositions that do not require any further preparation for patient application are simpler and very interesting.

  Liquid pharmaceutical formulations of interferon-β are described, for example, in WO 95/31213 and WO 2004/096263.

  WO 2005/084303 describes an interferon-beta 1b polymer composite composition.

  US Pat. No. 6,531,122 relates to pegylated interferon variants in a formulation combined with excipients, solubilizers, and buffers.

  WO 2004/060299 provides a method for the synthesis of cytokine polymer complexes comprising interferon-beta and its receptor binding antagonists.

  US 20060051320A1 relates to a lyophilized formulation of pegylated interferon using trehalose as a cryoprotectant.

  WO 99/48535 provides a formulation that prevents loss and damage of PEG-interferon alpha conjugates during and after lyophilization.

  WO 03002152 relates specifically to stabilized compositions comprising interferon polypeptides and sulfoalkyl ether cyclodextrin derivatives.

  None of the above references disclose or suggest the liquid formulation of the present invention.

SUMMARY OF THE INVENTION According to one embodiment of the present invention, a liquid pharmaceutical composition comprising pegylated interferon beta (PEG-IFN-β), an excipient, a surfactant, and a buffer, wherein the excipient is a polyol. Wherein the surfactant is a nonionic surfactant and the buffer is a sodium acetate buffer.

  In accordance with another aspect of the invention, for producing a liquid pharmaceutical composition comprising adding a calculated amount of excipients and surfactant to a buffer solution followed by addition of PEG-IFN-β. A method is provided.

  In accordance with another aspect of the present invention, there is provided a container sealed under conditions that are sterile and suitable for storage prior to use, comprising the liquid pharmaceutical formulation of the present invention.

  In accordance with another aspect of the present invention, there is provided a kit of pharmaceutical composition comprising a container filled with the pharmaceutical composition of the present invention.

FIG. 1 shows the% recovery, expressed as% recovery at AF and T0.

BRIEF DESCRIPTION OF TABLES AND FIGURES Formulation of the present invention comprising 0.044 mg / ml PEG-IFN-beta

  Table 1 shows various formulations of the present invention containing 0.044 mg / ml PEG-IFN-beta.

  Table 2 shows the stability test (SE-HPLC) over time of the formulations of the invention at pH 4.2 at 40 ° C, 25 ° C and 2-8 ° C, respectively.

  Table 3 shows the stability test (RP-HPLC) over time of the formulations of the invention at pH 4.2 at 40 ° C., 25 ° C. and 2-8 ° C., respectively.

  Table 4 shows the titer (mcg / mL) of the formulations of the present invention by SE-HPLC.

  Table 5 shows the titer (mcg / mL) of the formulation of the present invention by SE-HPLC before and after filtration using% recovery and the graph (Figure 1: AF and T0 recovery in%). % Recovery).

  Table 6 shows the pH values of the formulations of the present invention at 25 ° C. and 2-8 ° C. for 4 to 26 weeks, respectively.

  Table 7 shows the bioassay results over time for the formulations of the present invention.

  II. Formulations of the invention comprising 0.055 and 0.110 mg / ml PEG-IFN-beta, respectively:

  In these formulations, the amount of PEG-IFN-beta was increased to 0.055 and 0.110 mg / ml, respectively.

  Table 8 shows an SE-HPLC test that measures the formulation purity of the present invention over time at pH 4.2 at each of 40 ° C, 25 ° C, and 2-8 ° C.

  Table 9 shows a SE-HPLC test that measures the protein content of the formulations of the present invention over time at pH 4.2 at each of 40 ° C, 25 ° C, and 2-8 ° C.

  Table 10 shows an RP-HPLC test that measures the formulation purity of the present invention over time at pH 4.2 at each of 40 ° C, 25 ° C, and 2-8 ° C.

  Table 11 shows the pH values over time of the formulations of the present invention at 4-13 weeks at 25 ° C and 2-8 ° C, respectively.

  Table 12 shows the bioassay data for the formulations of the invention at 25 ° C and 2-8 ° C, respectively.

  Table 13 shows data relating to oxidized forms by peptide mapping of PEG-IFN-beta of the formulations of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is a liquid pharmaceutical composition comprising pegylated interferon beta (PEG-IFN-β, PEG-IFN-beta), an excipient, a surfactant and a buffer, said excipient Relates to the composition, wherein the agent is a polyol, the surfactant is a nonionic surfactant, and the buffer is a sodium acetate buffer.

  In one embodiment, the present invention provides a liquid pharmaceutical composition comprising PEG-IFN-β or a mutein thereof or an active fragment thereof, an excipient, a surfactant and a buffer, wherein the excipient is a polyol. And wherein the surfactant is a nonionic surfactant and the buffer is a sodium acetate buffer.

  In the following paragraphs, definitions of the various compounds that make up the formulations of the invention are given, and they are intended to apply throughout the specification and claims, unless a broader definition is provided elsewhere. .

  The pegylated interferon-beta of the formulation can be any interferon-beta that results from covalent modification of polyethylene glycol, or equivalent modification. One example of PEG-interferon-beta is PEG-interferon-beta, which can be PEGylated by known methods, such as those described in WO 99/55377.

  In particular, according to the present invention, IFN-beta is covalently linked to the hydrophobic polymer polyethylene glycol (PEG), also known as polyethylene oxide (PEO). The PEG can be a linear polymer with hydroxyl groups at each end:

  It can also be used as methoxy-PEG-OH (m-PEG), in which one end is a relatively inert methoxy group and the other end is a hydroxyl group that is chemically modified. :

  In the above formula, n may be 1 to several hundred.

  In another preferred embodiment, the PEG can also be a branched PEG represented by R (PEG-OH) m, where R is a central core, such as pentaerythritol or glycerol, and m is Indicates the number of branched arms. The branched arm (m) can range from 3 to 100 or hundreds. The hydroxyl group can be chemically modified.

Another form of branching of the invention is described, for example, in WO 96/21469, where the PEG has a single terminus that is chemically modified. This PEG form may be represented as (CH ₃ OPEG) pRX, where p is 2 or 3, R represents the central core, eg lysine or glycerol, and X is a functional group, eg chemically activated Represents a carboxyl formed.

  Another form of branching of the invention is designated as “pendant PEG” and it has a reactive group, eg carboxyl, along the PEG backbone rather than the end of the PEG chain.

  Furthermore, it is possible to produce PEG-IFN-beta of the present invention having weak or degradable linkages in its backbone as described, for example, in US patent application Ser. No. 06 / 026,716. Thus, PEG can be produced with ester linkages that undergo hydrolysis in the polymer backbone. According to the following scheme, the hydrolysis causes cleavage of the polymer into smaller molecular weight fragments:

  Copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and according to the present invention they can be used in place of PEG.

  Various methods have been developed for PEGylating proteins. PEG can be coupled to reactive groups found in proteins, usually utilizing electrophilically activated PEG derivatives. The α-amino group or the ε-amino group found in lysine residues, and the N-terminus resulting in a complex where the N-terminus consists of a mixture of products can be used.

  The complex preferably consists of a group of complexes bound to PEG molecules in the range of 1 to the number of amino acids in the protein per protein molecule.

  See, for example, Wohhiren et al., For the synthesis of thiol selective PEG derivatives. Bioconjugate Chem. , 4 (5): 314-318, 1993, it is preferred to introduce sites intended to be PEGylated into IFN-beta.

  In one embodiment, the IFN is specifically PEGylated. Specific PEGylation can be carried out according to the EP 675201 patent, for example at the N-terminal residue with mPEG-propionaldehyde. Woghiren et al. , Bioconjugate Chem. , 4 (5): 314-318, 1993 is particularly preferred.

An embodiment of the present invention is IFN-beta that is covalently attached to Cys ¹⁷ and monoPEGylated as described in WO 99/55377. The PEG moiety is a linear or branched methoxy PEG, hydrolytically or enzymatically degraded PEG, and one or more polyols and a copolymer of PEG and PLGA (poly (lactic acid / glycolic acid)). obtain.

  The thiol group of cysteine in embodiments of the present invention can be reacted with a PEGylation reagent that reacts with thiol. It can be a PEG containing functional groups such as orthopyridyl disulfide, vinyl sulfone, maleimide, iodoacetimide, for example. Preferably, the thiol-reactive PEGylation reagent is an orthopyridyl disulfide (OPSS) derivative of PEG. PEGylation reagents are used in a monomethylated form where only one end is utilized for conjugation or in a bifunctional form where both ends are utilized for conjugation.

In the reaction of a preferred embodiment, IFN-β-Cys ¹⁷ is used and it reacts according to the reaction scheme described herein. This reaction is site specific for Cys ¹⁷ because the other two cysteine residues (31 and 141) in IFN-β form a disulfide bond and therefore cannot be PEGylated. is there. PEG-IFN-β has an effective size comparable to the size of a protein having a molecular weight of 50-110 kDa, preferably about 70 kDa. In site-specific IFN-β modifications, the PEG molecules attached preferably have a molecular weight greater than 20 kD. In one embodiment, the PEG molecule attached to Cys ¹⁷ via a spacer is 2 × 20 kDa.

  The spacer and IFN form IFN 2 kDa-OPSS according to the following synthetic scheme.

  After PEGylation, the solution is purified to separate PEG-IFN-β from the free spacer and / or PEG. Preferably, this purification step is performed by ultrafiltration. Ultrafiltration is carried out at basic conditions below 10 ° C., preferably at a temperature of 5 +/− 3 ° C., more preferably at 4 ° C. In the purification step, preferably 0.1 M NaOH is used. The resulting PEG-IFN-β solution contains less than 0.5 EU / ml endotoxin, preferably less than 0.25 EU / ml endotoxin.

  In one embodiment, PEGylation is introduced into IFN-β according to the following scheme:

  In one embodiment, the low molecular weight PEG moiety can be conjugated with IFN-β.

  The low molecular weight PEG moiety has the formula:

[Wherein W and X are independently a group that reacts with an amine, sulfhydryl, carboxyl, or hydroxyl group, and binds the low molecular weight PEG moiety to IFN-β]
Have W and X are preferably independently selected from orthopyridyl disulfide, maleimide, vinyl sulfone, iodoacetamide, amine, thiol, carboxyl, active ester, benzotriazole carbonate, p-nitrophenol carbonate, isocyanate, and biotin. .

  The low molecular weight PEG moiety preferably has a molecular weight of 100 to 5000 daltons. In preferred embodiments, it is 1000 to 3000 daltons, preferably 1500 to 2000 daltons, and more preferably 2000 daltons.

  The monofunctional or bifunctional PEG moiety for attachment of the free end of the low molecular weight PEG to INF-β preferably has a molecular weight in the range of about 100-200 Daltons. It is preferably methoxy PEG, branched PEG, hydrolyzed or enzymatically degraded PEG, pendant PEG or dendrimer PEG.

  Monofunctional or bifunctional PEG is further represented by the following formula:

[Where:
Y is somewhat reactive at the terminal group on the free end of the low molecular weight PEG moiety that binds to IFN-β, and Z is reactive to the extent that it forms -OCH ₃ , or a bifunctional complex. It is a group that has]
Have

  Thus, in a step-wise manner, two or more PEG moieties are used to produce PEG-IFN-β that is useful in the compositions of the invention and for pharmaceutical use.

  Advantageously, the disulfide bond between PEG and IFN-β is stable in the blood circulation and is broken upon entry into the cell.

  PEG-IFN-β used in the formulations of the present invention has approximately the same or higher interferon-β activity compared to natural interferon-β.

  The excipient can be any polyol and, together with the other ingredients, results in a stable PEG-interferon-beta formulation. Examples of polyols are mannitol (PEARLITOL (R)), sorbitol (NEOSORB (R)), maltitol (MALTISORB), xylitol (XYLISORB), and maltitol (LYCASIN). A preferred polyol excipient is mannitol.

  In accordance with the present invention, nonionic surfactants are used. Nonionic surfactants include polyol derivatives, polyoxyethylene esters and ethers, poloxamers, nonylphenyl ether, polyvinyl alcohol, propylene glycol diacetate, alkanolamides. The nonionic surfactant may preferably be a poloxamer. Poloxamer 188 is particularly preferred.

  Any buffer that can maintain the pH at a selected level can be used. The buffer can be sodium acetate buffer or PBS. Sodium acetate buffer is particularly preferred.

  In one embodiment, the present invention provides a liquid pharmaceutical composition comprising PEG-interferon-beta, an excipient, a surfactant, and a buffer, wherein the excipient is mannitol, and the surfactant Is the poloxamer 188, and the buffer is a sodium acetate buffer.

  Interferon-beta can be naturally occurring human interferon-beta or recombinantly produced that is PEGylated according to the present invention. Furthermore, the interferon-beta (IFN-β) of the present invention is a glycoprotein produced in the body in response to viral infection.

  Interferon units or international units of interferon (U or IU in international units) are reported as a unit of measure of IFN activity, defined as the amount required to protect 50% of cells against viral damage. ing. An assay that can be used to measure biological activity is the reported cytopathic effect inhibition assay (Rubinstein, et al. 1981; Familyletti, PC, et al., 1981). In this antiviral assay for interferon, 1 unit / ml of interferon is the amount necessary to produce a 50% cytopathic effect. The units were determined by the Hu-IFN-beta international reference standard (Pestka, S. 1986) provided by the National Institutes of Health.

  Interferons are also referred to as biological response modifiers (BRMs) because they have an effect on the response of an organism to a tumor and affect recognition through immunomodification.

  Human fibroblast interferon (IFN-β) has antiviral activity and can also stimulate natural killer cells against neoplastic cells. It is an approximately 20,000 Da polypeptide induced by viruses and double-stranded RNA. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of the fibroblast interferon gene by recombinant DNA technology (Delynk et al. 1980). It is 166 amino acids.

  Rebif® (Merck Serono-recombinant human interferon-β) in multiple sclerosis (MS) interferon therapy is an interferon, (IFN) -beta-1a, produced from a mammalian cell line. Its recommended international common name (INN) is “interferon β-1a”.

  As used herein, “interferon beta” or “IFN-β” is any molecule defined as such in the literature, including, for example, any type of IFN-β referred to in the section above. It is intended to include IFN-β that is suitable according to the present invention is commercially available, for example as Rebif® (Merck Serono), Avonex® (Biogen Idec), as long as they show a binding moiety for a specific PEGylation. According to the invention, the use of human-derived interferon beta is likewise preferred. The term “interferon beta” as used herein is intended to include salts, functional group derivatives, variants, analogs and active fragments thereof.

  The term “interferon-beta (IFN-β)” as used herein, as obtained by isolation from biological fluids or as obtained by DNA recombination techniques from eukaryotic host cells, It is specifically intended to include human-derived fibroblast interferon and salts, functional group derivatives, mutants, analogs and active fragments thereof. Preferably, IFN-beta is intended to mean interferon beta-1a.

The term “mutein” as used herein differs in one or more amino acid residues of native IFN-β without significantly changing the activity of the resulting product compared to wild-type IFN-β. It is an analog of IFN-β and PEG-IFN-β that is substituted or deleted with an amino acid. These muteins are produced by known synthetic methods and / or by site-directed mutagenesis or by any other suitable technique. Preferred muteins include, for example, those described by Shepard et al. (1981), or Mark et al. (1984). In particular, the muteins of the present invention contain Cys ¹⁷ , although any of the above changes may be present in the molecule.

  Any such mutein preferably has an amino acid sequence sufficiently overlapping with that of IFN-β to have substantially the same or stronger activity than that of IFN-β. The biological functions of interferons are well known to those skilled in the art, and biological standards are established and available, for example, at the National Institute for Biological Standards and Control (http: // immunology organ / links / NIBSC) .

  Bioassays for the determination of IFN-β / PEG-IFN-β activity have been described in the literature. The IFN assay can be performed, for example, as described by Rubinstein et al., 1981. Thus, by routine experimentation, it can be determined whether any given mutein has substantially the same or stronger activity than IFN-β.

  The IFN-β and PEG-IFN-β muteins that can be used according to the present invention, or nucleic acids encoding them, are based on the techniques and guidance described herein without undue experimentation by those skilled in the art. A finite sequence substantially corresponding to the substituted peptide or polynucleotide normally obtained.

  Preferred changes for muteins of the present invention are what are known as "conservative" substitutions. Conservative amino acid substitutions of a polypeptide or protein of the invention can include synonymous amino acids in a group having sufficiently equivalent physicochemical properties, where substitutions between members of the group are related to the biology of the molecule. Will preserve the function. Amino acid insertions and deletions also include amino acids such as cysteine residues, particularly where the insertion or deletion is for a small number of amino acids such as less than 30, preferably less than 10, and is important for functional structure Obviously, in the case where they are not removed or replaced, they can be made in the sequences defined above without changing their function. Proteins and muteins produced by such deletions and / or insertions are within the scope of the present invention.

  Examples of the production of amino acid substitutions of proteins that can be used to obtain PEG-IFN-β muteins for use in the present invention are described by Mark et al., US Pat. No. 4,959,314, 4,588. , 585, and 4,737,462; by Koths et al., US Pat. No. 5,116,943, by Namen et al., US Pat. No. 4,965,195; by Chong et al., U.S. Pat. No. 4,879,111; and any known steps as described by Lee et al. In U.S. Pat. No. 5,017,691, and U.S. Pat. No. 4,904,584. Including lysine-substituted proteins described in (Shaw et al.). Specific muteins of IFN-beta are described, for example, in Mark et al., 1984.

  As used herein, “functional group derivatives” include derivatives of PEG-IFN-β, as well as their muteins and fusion proteins, and as a side chain at the residue or N-terminal group or C-terminal group. From the resulting functional groups, as long as they can be prepared by methods known in the art and they are pharmaceutically acceptable, i.e. they do not destroy the activity of proteins that are substantially equivalent to the activity of IFN-β, And unless a toxic characteristic is provided to the composition containing it, it is included in this invention. These derivatives include, for example, N-acyl of a free amino acid of an amino acid residue formed with an aliphatic ester of a carboxyl group, ammonia or an amide of a carboxyl group by reaction with a primary or secondary amine, an acyl moiety. Derivatives (eg, alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl groups (eg, of ceryl or threonyl residues) formed with an acyl moiety.

  As “active fragments” of PEG-IFN-β, muteins, and fusion proteins, the present invention relates to protein molecules alone or related molecules or residues that bind to them, such as sugar or phosphate residues, or protein molecules, Or any fragment or precursor of a polypeptide chain with an aggregate of its own sugar residues, provided that the fragment does not have a significant decrease in activity compared to the corresponding IFN.

  The term “salt” as used herein refers to both salts of the carboxyl group and to the acid addition salt of the amino group of the protein or analog thereof. Salts of carboxyl groups are formed by means known in the art and include inorganic salts such as sodium, calcium, ammonium, iron, zinc and the like and salts with organic bases such as triethanol Includes those formed using amines, arginine or amines such as lysine, piperidine, procaine. Acid addition salts include, for example, salts with mineral acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid or oxalic acid. Of course, any such salt must retain the biological activity of the protein (IFN) according to the present invention, such as the ability to bind to the corresponding receptor and initiate receptor signaling.

  In a further embodiment, the fusion protein included in the composition of the invention comprises an Ig fusion. The fusion may be linked directly or via a short linker peptide, which may be 1-3 amino acid residues or longer, for example 13 amino acid residues. The linker is introduced between an IFN sequence and an immunoglobulin sequence, for example, a tripeptide of EFM (Glu-Phe-Met), or Glu-Phe-Gly-Ala-Gly-Leu-Val- It can be Leu-Gly-Gly-Gln-Phe-Met. The resulting fusion protein may have improved properties, such as increased residence time in biological fluids, increased specific activity, increased expression levels, or enhanced purification of the fusion protein.

  Preferably, PEG-IFN-beta is about 0.1 mg / ml to about 0.01 mg / ml, preferably about 0.06 mg / ml to about 0.03 mg / ml, and more preferably about 0.044 mg / ml. Present in the composition at a concentration of

  In another embodiment, PEG-IFN-beta is present in the formulations of the invention at a concentration of 0.05 to 0.150 mg / ml, preferably at a concentration of 0.055 or 0.110 mg / ml.

  The dosage used in the composition, and the dose that can be applied to an individual, includes pharmacokinetic properties, route of administration, patient status and characteristics (sex, age, weight, health status, size), degree of symptoms, It will vary depending on a variety of factors, including combination therapy, frequency of treatment, and desired effect.

  Standard dosages of human IFN-beta / PEG-IFN-beta range from 80000 IU / kg to 200000 IU / kg per day, or 6 MIU (million international units) per person per day to 12 MIU, or 22-per person Within the range of 44 μg (microgram) IFN-beta equivalent. In accordance with the present invention, PEG-IFN-β in the compositions of the present invention is preferably about 1-50 μg per person per day, more preferably about 10-30 μg, or about 10-20 μg per day, preferably in terms of IFN-beta. Can be used in dosages.

  Administration of the active ingredients of the present invention can be by intravenous, intramuscular, or subcutaneous routes. Preferred routes of administration of the compositions of the present invention are the subcutaneous route and the intramuscular route.

  The PEG-IFN-β compositions of the invention can also be administered daily, every other day, or less frequently. Preferably, the PEG-IFN-β composition is administered once, twice or three times per week. Preferably it is administered once every two weeks.

  A preferred route of administration is subcutaneous administration, for example, three times a week. A further preferred route of administration is intramuscular administration, which can be administered, for example, once a week.

  Preferably, 22-44 [mu] g, or 6 MIU-12 MIU of PEG-IFN-beta in the composition of the present invention in terms of IFN-beta is administered three times a week by subcutaneous injection.

  The PEG-IFN-beta composition can be administered subcutaneously every other day at a dosage of 25-30 μg, or 8 MIU to 9.6 MIU. 30 μg in terms of IFN-beta, or 6 MIU of PEG-IFN-beta can also be administered intramuscularly once a week.

  The term “stability” refers to the physical, chemical, and structural stability of the interferon formulations of the present invention (including the maintenance of biological effects). Larger order polymer formation, deglycosylation, glycosylation modification, oxidation, or any other that reduces at least one biological activity of an interferon polypeptide included in the present invention by chemical degradation or aggregation of protein molecules This structural modification can cause instability of the protein formulation.

  A “stable” composition, solution, or formulation refers to one in which the degree of protein degradation, modification, aggregation, loss of biological activity, etc. is acceptable controlled and does not increase unacceptably over time. . Preferably, the formulation has a labeled interferon activity of at least 60% or about 60%, more preferably at least 70% or about 70%, most preferably at least 80% or about 80% of 12-24 months. Maintained over. Preferred PEG-IFN-β compositions of the present invention, when stored at 2-8 ° C., are at least about 6 months, 12 months, 18 months, more preferably at least about 20 months, even more Preferably it has a shelf life of at least about 22 months, most preferably at least about 24 months.

  Methods for measuring the stability of the PEG-IFN-β liquid pharmaceutical compositions of the present invention are available in the art, including the methods described herein. Thus, PEG-IFN-β aggregate formation during storage of the liquid composition of the present invention can be readily determined by measuring changes in dissolved PEG-IFN-β in solution over time. The amount of dissolved polypeptide in solution can be quantified by a number of analytical assays applied to the detection of specific PEG-IFN-β. Such assays include, for example, reverse phase (RP) -HPLC and UV absorption spectroscopy as described in the examples below.

  To distinguish between soluble polypeptide fractions present as soluble aggregates and fractions present in non-aggregating biologically active molecular forms, for example as described in the examples below. Using simple analytical ultracentrifugation, determination of both soluble and insoluble aggregates during storage in a liquid composition can be achieved.

  The term “buffer” or “physiologically acceptable buffer” is known to be safe for pharmaceutical or veterinary use in a formulation and the pH of the formulation is suitable for the formulation. It is a solution of a compound having the effect of maintaining or adjusting in a wide pH range. Buffers acceptable for adjusting pH at a moderately acidic pH include, but are not limited to, compounds such as phosphate, acetate, citrate, arginine, TRIS, and histidine. “TRIS” refers to 2-amino-2-hydroxymethyl-1,3-propanediol and any pharmaceutically acceptable salt thereof. A preferred buffer is saline or an acetate buffer with an acceptable salt.

  The excipient is preferably present at a concentration of about 30 mg / ml to about 50 mg / ml, more preferably at a concentration of about 40 mg / ml to about 50 mg / ml, even more preferably at a concentration of about 45 mg / ml. .

  The surfactant is preferably at a concentration of about 0.1 mg / ml to about 1 mg / ml, preferably at a concentration of about 0.4 mg / ml to about 0.7 mg / ml, even more preferably about 0.5 mg. / Ml concentration.

  The term “surfactant” refers to a soluble compound that reduces the surface tension of a liquid, or reduces the interfacial tension between two liquids, or between a liquid and a solid, where surface tension is the surface area. Is the force acting on the liquid surface that tends to minimize.

  Surfactants are sometimes used in pharmaceutical formulations, including the delivery of low molecular weight drugs and polypeptides, for altered drug absorption or delivery to target tissues. Well known surfactants include polysorbates (polyoxyethylene derivatives of sorbitol fatty acid esters; Tween®), and poloxamers such as Pluronic® or Lutrol marketed by BASF (Germany). ) (Registered trademark).

  In accordance with a preferred embodiment of the present invention, a surfactant selected from Pluronic® F77, Pluronic® F87, Pluronic® F88, and Pluronic® F68, particularly preferably Pluronic®. (Trademark) F68 (BASF, Pluronic® F68, also known as Poloxamer 188) is used to formulate PEG-IFN-β to produce vials and / or delivery devices (eg, syringes, pumps) It has been found that a stable formulation is obtained that minimizes the loss of the source of activity caused by absorption on the surface of the catheter, etc.). A surfactant selected from Pluronic® F77, Pluronic® F87, Pluronic® F88, and Pluronic® F68, particularly preferably Pluronic® F68 (BASF, Pluronic ( Stable composition that is more resistant to oxidation and formation of protein aggregates by formulating PEG-IFN-β using (registered trademark) F68, also known as Poloxamer 188) Has also been found to be obtained.

  Pluronic® surfactant is a block copolymer of ethylene oxide (EO) and polyethylene oxide (PO). The propylene oxide block (PO) is sandwiched between two ethylene oxide (EO) blocks.

  In Pluronic® F77, the percentage of polyoxyethylene (hydrophilic material) is 70% and the molecular weight of hydrophobic material (polyoxypropylene) is about 2,306 Da.

  In Pluronic® F87, the percentage of polyoxyethylene (hydrophilic material) is 70% and the molecular weight of hydrophobic material (polyoxypropylene) is about 2,644 Da.

  In Pluronic® F88, the percentage of polyoxyethylene (hydrophilic material) is 80% and the molecular weight of hydrophobic material (polyoxypropylene) is about 2,644 Da.

  In Pluronic® F68, the percentage of polyoxyethylene (hydrophilic material) is 80% and the molecular weight of hydrophobic material (polyoxypropylene) is about 1,967 Da.

  Other polymers having properties comparable to those of the Pluronic series can also be used in the compositions of the present invention. Preferred surfactants are Pluronic® F68 and surfactants with similar properties.

  Pluronics, especially Pluronic® F68, is used at concentrations that are sufficient to maintain the stability of PEG-IFN-β for the desired storage period (eg, 12-24 months), as well as, for example, vials Preferably present at a concentration that is sufficient to prevent protein loss due to absorption on the surface of the ampule, or cartridge or syringe.

  Preferably, the concentration of Pluronic®, especially Pluronic® F68, in the liquid formulation is 0.01 or about 0.01 mg / ml to 10 or about 10 mg / ml, more preferably 0.05 or about 0. 0.05 mg / ml to 5 or about 5 mg / ml, particularly preferably 0.1 or about 0.1 mg / ml to 2 or about 2 mg / ml, most preferably 1 or about 1 mg / ml.

  In another preferred embodiment, methionine, particularly L-methionine is used. A concentration of 0.1 mg / ml to 0.5 mg / ml, preferably about 0.1 mg / ml to 0.3 mg / ml, more preferably about 0.25 mg / ml or about 0.12 mg / ml is particularly useful. .

  It has been found that the addition of methionine advantageously further stabilizes the formulation and reduces protein oxidation.

  The various compounds contained or included in the formulations of the present invention can be varied as described above and the favorable effects of methionine can be achieved. In one embodiment, the formulation is 0.055 mg / ml PEG-IFN-beta, about pH 3.5-4.5, preferably pH 4.2 10 mM sodium acetate buffer, 45 mg / ml mannitol, and 0 Contains or contains 5 mg / ml poloxamer 188, or 0.110 mg / ml PEG-IFN-beta.

  The composition of the present invention comprises a sufficient amount of buffer to maintain the pH of the composition within plus or minus 0.5 units of the specified pH, wherein the specified pH is about 3. 0 to about 5.0, preferably about 3.5 to 4.5, and even more preferably about 4.2 ± 0.2.

  The buffer is present in the composition of the present invention at a concentration of about 5 mM to 500 mM, preferably about 10 mM.

  The composition is preferably an aqueous solution.

  The present invention includes a liquid composition. A preferred solvent for injection is water.

  Adjusting the pH to about pH 3-5, preferably 3.5-4.5, and more preferably about 4.2 +/− 0.2 for PEG-IFN-β provides a stable formulation. Can be shown.

  The composition of the present invention has achieved positively affecting the degradation process of PEG-IFN-β, which can be negatively affected in the liquid composition. This effect can be measured by SE-HPLC, for example at 40 ° C. and 25 ° C., respectively. The composition of the present invention was obtained without a significant decrease in PEG-IFN-β content observed (by SE-HPLC). In particular, no decrease in PEG-IFN-β content was detected up to 2 weeks at 40 ° C. and 6 weeks at 25 ° C. The composition of the present invention exhibits good stability properties and in particular a reduction of protein aggregation during storage can be achieved.

  0044 mg / ml PEG-IFN-β, 10 mM sodium acetate at about pH 4.2, 45 mg / ml, prepared in a glass vial, preferably 3 ml, or in a glass syringe, preferably 1 ml. Positive results can be achieved in particular for compositions comprising mannitol and 0.5 mg / ml poloxamer 188.

  The composition of the present invention has excellent stability as measured by SE-HPLC, purity using RP-HPLC, excellent protein content as measured by SE-HPLC, and excellent biological Shows activity.

  In another aspect, the invention provides a method for preparing the above liquid pharmaceutical composition, wherein a calculated amount of excipients and surfactant is added to the buffer, followed by PEG-IFN-β. To the process.

  In yet another aspect, the present invention relates to a sealed container containing the liquid pharmaceutical formulation of the present invention under conditions that are sterile and suitable for storage prior to use.

  Any container that is suitable for pharmaceutical applications can be used. The container is preferably a pre-filled syringe, vial or cartridge for a self-injector.

  The formulations of the invention can be administered using the evaluated device. Examples including these single vial systems include self-injection devices or pen-type injection devices for solution delivery, such as Rejectect®.

  The product claimed in this application includes packaging material. The packaging material provides the conditions under which the product can be used in addition to the information required by the governing body. If necessary, the packaging material of the present invention provides instructions to the patient in order to prepare a final solution and to use such a final solution for more than 24 hours in two vials, wet / dry products. provide. For single vial solution products, the label indicates that such a solution can be used for a period of 24 hours or more. The product claimed herein is useful for use in human pharmaceutical products. The composition can be provided to the patient as a clear solution.

  PEG-IFN-β is in accordance with the present invention SC or IM injection, transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micropump, oral, or other means recognized by those skilled in the art. It can be administered to a patient via various delivery methods, including:

  In another aspect, the present invention relates to a pharmaceutical composition kit comprising a container filled with the pharmaceutical composition of the present invention.

  The container is preferably a syringe for use as a delivery device.

  All references cited herein, including journal articles or abstracts, published or unpublished U.S. or foreign patent applications, issued U.S. or foreign patent applications or any other references, All data, tables, figures, and text in the cited references are hereby fully incorporated by reference. Moreover, the entire contents of the references cited in the references cited herein are likewise fully incorporated by reference.

  In the following, the invention is illustrated by examples, which are not to be construed as limiting the scope of the invention.

Embodiments of the invention will be illustrated by the following examples.
1. Production of PEG-IFN-β Complex An example of the production of PEG-IFN-β is shown in the following scheme:

2. Formulation The PEG-IFN-β composition was formulated using PEG-IFN-beta 1a. The solution was filtered through a 0.22 um membrane (Durapore) and filled into a final container (1 ml in a vial or syringe).

  The test composition contained 0.044 mg / ml PEG-IFN-beta1a, 10 mM sodium acetate buffer pH 4.2, 45 mg / ml mannitol, and 0.5 mg / ml poloxamer 188. Alternatively it contained 0.055 or 0.110 mg / ml PEG-IFN-beta 1a and optionally methionine.

  Samples of the test composition are stored at 40 ° C., 25 ° C. and 2-8 ° C., respectively, and for determination of purity by SE-HPLC or RP-HPLC, for determination of protein content by SE-HPLC Tests for IFN-beta-induced cell protection and determination of biological activity by pH-based antiviral assays over time and for determination of oxidized forms by peptide mapping / UPLC analysis went.

3. Stability assay and other experiments 3.1 Purity and assay by SE-HPLC The purity by SE-HPLC was evaluated on a Shodex column (aqueous SE, code KW-803); 1.0 mL / min in isocratic mode Elution from PBS 10x (Gibco BRL code 70013-016) was performed using PBS 1x prepared by diluting 10 times with water; detection was performed at 214 nm UV.

  IFN-PEG formulated samples were injected using the following injection volumes: 200 mcL (for 44 and 55 mcg / mL samples); 100 mcL (for 110 mcg / mL samples).

  For the assay, protein quantification was performed against the reference standard PS200-01 (single point assay).

3.2 Purity by RP-HPLC Purity by RP-HPLC is performed on a C4 column (Symmetry 300 C4, 5m size 4.6 x 250 mm, Waters), temperature adjusted to 35 ° C; wavelength set to 214 nm, and Elution was performed at 1 mL / min using the following conditions:

  IFN-PEG formulated samples were injected using the following injection volumes: 160 mcL (for 44 mcg / mL sample); 200 mcL (for 55 mcg / mL sample); 100 mcL (for 110 mcg / mL sample).

3.3 Biological activity (in vitro bioassay)
Biology using methods available for IFN-β, an antiviral assay based on IFN-β-induced protection of cells (WISH cells-human amniotic tissue) against the cytopathic effect of viruses (Vesicular Stomatis Virus) Activity was measured.

3.4 Determination of pH The pH measurement was performed according to standard operating procedures using a calibrated pH meter (Mettler-Toledo, mod. 713).

3.5 Oxidized Forms by Peptide Mapping / UPLC Methods for quantification of oxidized methionine residues (Met1, Met117, Met36) were performed by proteolysis of IFN-PEG samples with endoprotease Lys-C and subsequent gradient UPLC. Predict analysis; pre-treatment of the formulated sample is performed prior to proteolysis to remove any obstacles due to substrate components.

  Separation of the proteolytic mixture was carried out on an Acquity UPLC analytical column (BEH C18 1,7 μm 2.1 × 50 mm code. 1860002350, Waters); 0.1% TFA / water (A1) and 0.1% TFA / acetonitrile Elution was performed under gradient conditions using (B1).

  The following instrument settings and analysis parameters may be used:

Claims (25)

PEGylated interferon-β (PEG-IFN-β) , wherein the PEG has a molecular weight of at least 20 kDa, comprising the PEG-IFN-β , a polyol excipient, a poloxamer 188 surfactant, and a sodium acetate buffer A liquid pharmaceutical composition, wherein the pH is 4.2 ± 0.2.   The composition of claim 1, wherein the polyol excipient is mannitol.   The composition according to claim 1 or 2, wherein the PEG-IFN-β is present at a concentration of 0.01 mg / ml to 0.1 mg / ml.   4. The composition of claim 3, wherein the PEG-IFN- [beta] is present at a concentration of 0.044 mg / ml, 0.055 mg / ml, or 0.110 mg / ml.   The composition according to claim 1, wherein the PEG-IFN-β comprises linear or branched PEG.   The composition according to claim 1, wherein the PEG-IFN-β comprises a branched PEG.   The composition according to claim 5 or 6, wherein the PEG has a molecular weight of at least 40 kDa.   The composition according to claim 5 or 6, wherein the PEG has a molecular weight of 40 kDa.   The composition according to claim 1 or 2, wherein the polyol excipient is present in a concentration of 30 mg / ml to 50 mg / ml.   The composition according to claim 1 or 2, wherein the polyol excipient is present in a concentration of 40 mg / ml to 50 mg / ml.   The composition of claim 1 or 2, wherein the polyol excipient is present at a concentration of 45 mg / ml.   The composition according to claim 1 or 2, wherein the poloxamer 188 is present in a concentration of 0.1 mg / ml to 1 mg / ml.   The composition of claim 1 or 2, wherein the poloxamer 188 is present in a concentration of 0.4 mg / ml to 0.7 mg / ml.   The composition of claim 1 or 2, wherein the poloxamer 188 is present at a concentration of 0.5 mg / ml.   The composition according to claim 1 or 2, wherein the sodium acetate buffer is present at a concentration of 5 mM to 500 mM. The composition of claim 15 , wherein the sodium acetate buffer is present at a concentration of 10 mM. The composition according to any one of claims 1 to 16 , wherein the composition is an aqueous solution. The composition according to any one of claims 1 to 17 , wherein the composition further comprises methionine. 19. A composition according to claim 18 , wherein methionine is present at a concentration of 0.10 to 0.50 mg / ml. 19. The composition of claim 18 , wherein methionine is present at a concentration of 0.20-0.40 mg / ml. 19. A composition according to claim 18 , wherein methionine is present at a concentration of 0.12 mg / ml or 0.25 mg / ml. A method for producing a liquid pharmaceutical composition according to any one of claims 1 to 21 , wherein a calculated amount of polyol excipient and poloxamer 188 is added to the sodium acetate buffer solution, after which The method, wherein PEG-IFN-beta is added. A container that is sterile and sealed under conditions suitable for storage prior to use, comprising the liquid pharmaceutical composition according to any one of claims 1 to 21 . 24. A container according to claim 23 which is a pre-filled syringe or vial for a self-injector. A kit of a pharmaceutical composition comprising a container filled with the pharmaceutical composition according to any one of claims 1 to 21 .

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